فهرست مطالب پیمان رحیمی فیلی

Spermatogonial stem cells have the ability to self-renewal and differentiation and can transmit genetic traits to the next generation. Therefore, establishing an appropriate culture medium for this cell line is important. The aim of this study was to determine the effect of different concentrations of L-arginine on sheep spermatogonial stem cells colony formation in-vitro.

An experimental study was conducted. Each treatment was replicated five times. Spermatogonial cells were isolated from prepubertal lamb’s testis using two-step enzymatic digestion. The cells were cultured for ten days in six groups. In the control group, a simple culture of spermatogonial cells was performed in DMEM containing 1% antibiotics and 5% FBS. In the treatment groups 1, 2, 3, 4, and 5 different concentrations of L-arginine (50, 100, 200, 500, and 1000 μmol/L), was added to the culture medium, respectively. The culture media were changed every three days. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 and PLZF antigens. Immediately after isolation, the percentage of cells viability, number, and surface area of colonies formed on the 4th, 7th, and 10th days after the culture, were assessed by an inverted microscope. Data were analyzed using one- way ANOVA test. P<0.05 was considered statistically significant.

The findings indicated that the viability rate of spermatogonial cells after isolation was 89.28 ± 1.4%. Isolated cells expressed PGP9.5 and PLZF antigens. Also, on day 10 after culture and in treatment 4 (200 μmol/L L- arginine); the number (160.8 ± 21.1) and surface area (5.4 ± 1.3) spermatogonial cells colonies had a significant increase compared to other groups (P<0.05).

It seems that the addition of L- arginine at a dose of 200 μmol/L has a positive effect on the induction of spermatogonial stem cells colonization and can provide an appropriate culture medium in vitro.

In vitro maturation (IVM) of oocytes and subsequently, in vitro fertilization (IVF) for the generation of embryos in the laboratory have important values. Considering that antioxidants are known as effective free radicals scavenger, it is possible to improve the in vitro oocyte maturation and the fetal quality. Therefore, the aim of this study is to evaluate the effect of Thymbra spicata hydroalcoholic extract as a source of antioxidant on in-vitro sheep oocyte maturation. Cumulus oocyte complexes (COCs) were collected from ewe ovaries and were cultured for 24 hours in maturation medium in TCM supplemented with FSH, LH, FBS, cysteamine, pyruvate sodium and antibiotics (control group) and in maturation medium without cysteamine (as an antioxidant) supplemented with different doses of Thymbra spicata hydroalcoholic extract (1mg/ml: group 1, 10 mg/ml: group 2, 50 mg/ml: group 3) as an antioxidant. In-vitro maturation stages and resumption of meiotic was assessed by determination of cumulus cells mass expansion and number of oocytes in metaphase II stage of meiotic division in all groups. Cumulus cells mass expansion was similar between control, 2 and 3 groups. However, in group 1 was lower than control group. Nuclear maturation was similar between control and group 3 and both of them were different with groups 1 and 2. The results of this study showed that the Thymbra spicata hydro alcoholic extract, has a positive effect on oocyte maturation that is doses dependent. So with increasing concentration of Thymbra spicata hydroalcoholic extract, the rate of maturation immature oocytes is increased. Generally, we conclude that addition of appropriate amounts of natural extracts such as Thymbra spicata to maturation medium improves oocytes maturation.

Many previous studies have proved that the anatomical features of ewe cervix can affect the success of artificial insemination. These characteristics differ in sheep breeds. This study aimed to describe the anatomical features of cervix in Zandi ewes.One hundred and ninety threenonpregnant and clinically healthy reproductive tracts of adult and non-adult Zandi sheep were collected from a slaughter house and were divided into follicular or luteal phase.Then, the morphology of the vaginal protrusion of cervix was classified as slit, papilla, duckbill, flap or rose. The depth of penetration of an inseminating pipette was recorded. The cervical canal of each tract was filled with a silicone sealant for casting the mould. The cervices were sectioned longitudinally, and the length, number of cervical rings and the arrangement of the rings were recorded. The degree of completeness and interdigitations of the folds recorded as one of three grades 1, 2, and 3 cervices. The results showed the Papillatype was more common in vaginal protrusion of cervix and the most depth of penetration was in Slittype. The mean length of cervix, distance from cervix external os to first ring and the depth of penetration in adult ewes were significantly more than those in non-adult ewes. The mean distance betweencervical external osand first and second ringsand the depth of cervical penetration in follicular phase were more than those in luteal phase.The mean number of cervical ridges was 5 and 6 in 94% and 6% of cervices, respectively. Grades 1, 2, and 3 cervices, were observed in 64%, 25% and 11% of samples, respectively. The information generated in this study would be useful for increasing the success rate of penetration in ewes exhibiting estrus in order to improve the lambing rate of tropical ewes following transcervical AI.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, because they have the capacity of self-renewal, and differentiation into spermatozoa. Freezing is the most common long-term preservation approach for SSCs. The present study aimed to investigate the cryoprotective impacts of FBS (Fetal Bovine Serum) and trehalose on caprine SSCs.

SSCs were isolated from prepubertal goat testis by enzymatic digestion and differential plating. Cells were divided into 9 groups. The control group included SSCs without cryoprotective agents. In the treatment groups 1, 2, 3 and 4, concentration of 10% FBS, and various concentrations of trehalose (0, 50, 100 and 200 mM), and in the treatment groups 5, 6, 7, and 8, concentration of 20% FBS and various concentrations of trehalose (0, 50, 100 and 200 mM) were used respectively. The viability rate of the cells was assessed immediately after isolation, following addition of cryoprotectant agents and after thawing. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 antigen. Data were analyzed using one-way ANOVA test.

The viability rate of SSCs in various treatments following addition of FBS and trehalose were similar to viable cells immediately after isolation. Furthermore, higher viability rates of SSCs after thawing were observed in freezing medium containing 10% FBS and 200 mM trehalose (P<0.05).

The results revealed that freezing in 10% FBS with 200 mM trehalose acts as efficient method for the cryopreservation of caprine SSCs.

Spermatogonial stem cells are specific cells that have the ability of self-renewal and differentiation. These cells play an essential role in maintaining spermatogenesis and fertility. In this regard, the present study was performed with the purpose of investigating the effect of different concentrations of follicle stimulating hormone (FSH) on in vitro colony formation of caprine spermatogonial stem cells.

Spermatogonial cells, were isolated from prepubertal goat testis using two-step enzymatic digestion. Then, isolated cells were cultured for 10 days in four groups. In the control group, simple culture of spermatogonial cells was performed in Dulbecco's Modified Eagle Medium (DMEM) containing 1% antibiotics and 5% FBS (Fetal Bovine Serum). In the treatment groups 1, 2, and 3, different concentrations of follicle stimulating hormone (5, 10, and 20 IU/ml), was added to the culture medium, respectively. The culture media were changed every 72 hours. Identification of cells was confirmed by immunocytochemical staining against PGP9.5 antigen. Immediately after isolation, percentage of cells viability, surface area, and number of colonies formed on 4th, 7th and 10th days after the culture, were evaluated using an inverted microscope. Data were analyzed using one way ANOVA test.

The findings indicated that viability rate of Spermatogonial stem cells after isolation was 89.4 ± 2.32%. The effect of FSH on the formation of spermatogonial cells colonies was dose dependent. Doses of 5 and 10 IU/ml increased the surface area and number of the spermatogonial cell derived colonies but dose of 20 IU/ml reduced colonies formation (p < 0.05).

FSH can provide an appropriate culture medium for the study of spermatogonial cells in vitro.

Spermatogonial stem cells (SSCs) are undifferentiated germ cells that maintain spermatogenesis during lifetime by balancing between self-renewal and differentiation. The aim of this study was to investigate the effect of different concentrations of vitamin E on spermatogonial stem cells colony formation.

SSCs were isolated from testes of prepubertal lamb by two step enzymatic digestions then purified by differential pllating. The cells were cultured for 10 days in 4 groups. Control group: Basic media (DMEM environment which contains 1% antibiotic and 5% FBS( and treatment groups were treated with 20, 40 and 80 µmol/ml vitamin E added to basic media respectively. Changing of culture media was performed every 72h. Colony number and diameter assay was done by inverted microscope. Identification of SSCs was performed by immunocytochemistry assay against PGP9.5.

there were no significant difference in colony number and surface area between treatment groups and between treatment and control groups in days 4, 7and 10. Colony surface area was significantly increased at day 10 compared to day 4 (P<0.05).

The results of this study showed vitamin E as an antioxidant doesn’t have significant effect on colony induction of SSCs in short term culture in vitro.

The technology of using spermatogonial stem cells (SSCs) has been limited due to lack of an ideal culture system for growth and proliferation. The aim of this study was to investigate the effects of different doses of vitamin C on SSCs colony formation in vitro.
The cells were isolated from testes of prepubertal lambs by two enzymatic digestions, purified by differential platting, and then treated for 10 days by using 4 Simple culture including SSCs in DMEM containing 1% antibiotic and 5% FBS as our control group and for the three other cultures we used the same culture medium as that in control group plus 20, 40 and 60 µg/ml of vitamin C respectively. Culture media were refreshed every 72h and colony numbers and diameters were determined on the 4th , 7th and 10th days after the beginning of culture by using inverted microscope. Spermatogonial cells were identified by immunocytochemistry staining against PGP9.5. Using R software, the results obtained from 5 repeats were evaluated by ANOVA. P On the 7th day, we found a significant difference between the culture No. 2 (0.41 mm2) and culture No. 3 (0.08 mm2) in regard to spermatogonial colonies surface areas (P The results of this study showed that vitamin C with a dose of 40 (μg/ml) was effective in increasing the surface area of spermatogonial colonies. But it had no effect on spermatogonial cell number.

Spermatogonial stem cells (SSCs) are able to establish balance between self-renewal and differentiation, thereby maintain the spermatogenesis. The aim of this study was to investigate effects of different doses of Testosterone on SSCs colony formation.
The cells were isolated from testes of prepubertal lambs by two-step enzymatic digestion and then purified by differential platting. The cells were cultured for 13 days in 4 groups: Control [DMEM containing antibiotic (1%) and FBS (5%)]; and Treatment groups 1, 2 and 3 (DMEM䷫ⶢ쭞꺉 60, 120 and 240 µg/ml, respectively). The culture media was changed every 72 h. Identification of SSCs was performed by immunocytochemistry staining against PGP9.5. Finally, following the evaluation of percentage for viability rate of SSCs immediately after isolation and also the number and surface areas of the colonies on days 5, 9 and 13 after the beginning of culturing by invert microscopy, the analyses were made using statistical tests.
The percentage for viability rate of SSCs after isolation was 86.77±3.63%. The decrease in the number and surface areas of spermatogonial colonies in Treatment groups 2 and 3 were significant compared to Control and Treatment group1 (P The results of this study showed that Testosterone has no effect on colony induction of SSCs in vitro. Increasing the Testosterone dose decreases the colony number and surface area of SSCs.

Spermatogonial stem cells (SSCs) have diverse applications in reproductive medicine and biotechnology. Cryopreservation is the well-known method for long-term storage of these cells. The aim of this study was to investigate the effect of different concentrations of fetal bovine serum (FBS) and Sucrose on the viability rate of spermatogonial cells.
Testicular cells of pre-pubertal lambs were separated in a two-step enzymatic isolation and purified by differential plating. Then, the cells were divided in 6 groups. Groups 1, 2 and 3 were treated with FBS (10%) and Sucrose (0.07, 0.14 and 0.21 molar concentration (M) ) and groups 4, 5 and 6 with FBS (20%) and Sucrose (0.07, 0.14 and 0.21 M), respectively. The viability rate of the cells was evaluated immediately after isolation, addition of cryoprotectant agents and thawing procedures. Identification of spermatogonial cells in the culture was performed using the immunocytochemistry staining against PGP9.5.
The results showed that cryoprotectant do not have any harmful effects on lamb´s SSCs. Moreover, viability rate of the cells in freezing media containing FBS (10%) is significantly higher than the media containing FBS (20%). Furthermore, increasing concentrations of Sucrose (0.07, 0.14 and 0.21 M) had no beneficial effect on the spermatogonial viability rate.
It was concluded that freezing media containing dimethyl sulfoxide (10%) and FBS (10%) and Sucrose (0.07 M) is appropriate for cryopreservation of lamb spermatogonial cells.

Preparing a suitable condition for in vitro culture of spermatogonial stem cells can help discover all of the molecular mechanisms involving in spermatogenesis and pave the way for gene manipulation. This study aimed to evaluate the effect of a poly L-lactic acid (PLLA) nanofiber on the proliferation of frozen-thawed bovine spermatogonial stem cells in vitro. The isolated spermatogonial cells from the prepubertal bull were frozen-thawed and cultured in two groups: the control group (C) including the spermatogonial cells and the treatment group (T) including the spermatogonial cells seeded onto PLLA. The cells in both groups were cultured in DMEM supplemented with 5% fetal bovine serum and 40 ng/ml glial cell line-derived neurotropic factor (GDNF) for 2 weeks. Colony assay was conducted on days 4, 7, 10 and 13 after the beginning of the culture. Expression of spermatogonial genes (PLZF, BCL6, GFRα-1, VASA, Itgα6) in the culture was determined by reverse transcription polymerase chain reaction (RT-PCR) on the last day of the experiment. The viability rates of fresh cells before and after the addition of cryoprotectant agent were 87.4±2.4 and 81.8±3.1, respectively which decreased to 65±1.7 after the thawing (P<0.01). Moreover, the results showed that the surface area of colonies derived from spermatogonia were significantly greater in the treatment group compared to the control group on day 13 (P<0.05). Finally, RT-PCR revealed the expression of all spermatogonial stem cell (SSC) markers in both groups. The PLLA nanofiber can provide a suitable microenvironment for frozen-thawed SSCs in an in vitro-culture system.

Perosomus elumbis is an occasional congenital anomaly of cattle, swine, sheep, and dogs with unknown etiology. This congenital abnormality occurs in both sexes. A dead Holstein calf characterized by musculoskeletal and external genitalia abnormalities was referred to the large animal hospital of University of Tehran. Radiographic evaluation and subsequent dissection revealed that the vertebral column was truncated at the level of first lumbar vertebra (L1). Moreover, L2-L5, sacrum and coccygeal vertebrae were absent. The dorsum of the lumbosacral region contained only soft tissues. Urogenital tract was incomplete, and it contained agenesis of the ovaries, uterine tubes, cervix, and vulva concurrent with unilateral umbilical artery agenesis. Small and large intestine contained blind-ended sacs. No testes, scrotum, and penis were found. The intact ureter was attached to a thin-walled fluid fill sac. The laboratory finding showed that the pH of the fluid was 6 and contained hemoglobin, white blood cells, bacteria, a few red blood cells, oxalate crystalline, and epithelial cells. It was concluded that the collected fluid was urine. This is the first report of perosomus elumbis in a Holstein calf having a lot of visceral abnormalities in Iran.

Spermatogonial stem cells (SSCs) are undifferentiated cells with special capabilities on the self-renewing and differentiation. Proliferation of SSCs is a primary requirement for the study of their characteristics and function in vitro. The aim of this study was to evaluate the effects of different concentrations of follicle stimulating hormone (FSH) on the colonization activity of ovine SSCs in a short-term in-vitro co-culture with sertoli cells. Both sertoli and spermatogonial cells were isolated from 2-3 months old lamb testes by two-steps enzymatic digestion. Afterward، isolated cells were cultured in four groups with different concentrations of FSH (0، 5، 10 and 15 IU/ml، respectively) for 10 days. Colony assay (number and surface area) was evaluated 4، 7 and 10 days after the beginning of the culture by light microscope. At the day 4، colony surfaces of groups 1 and 2 were significantly more than groups 3 and 4 (P < 0. 05). At the days 7 and 10، colony surfaces of groups 1 and 2 were significantly more than group 4 (P < 0. 05). The results showed that treated groups with 10 and 15 IU/ml of FSH significantly decreased colony surface of ovine SSCs in comparison with co-culture system.

Spermatogonial stem cells (SSCs) are infrequent self-renewing cells among the type A spermatogonia within the seminiferous tubules and are the basis of spermatogenesis in mammalian testis. An adequate number of SSCs is a primary requirement for the study of their behavior، regulation، and further biomanipulation. In this paper، we studied the development of the primary co-cultures of type A spermatogonia and prepubertal bovine sertoli cells in the presence of Colony Stimulating Factor 1 (CSF1)، a potential contributor in the SSC niche. The effect of different concentrations of CSF1 (0، 10، 50 and 100 ng/mL) on the colonization activity of spermatogonial cells was assessed 4، 7 and 11 days after the beginning of the culture by counting the total number of colonies and measuring their area in each group of the present experiment. Immunofluorescent staining against OCT4 and vimentin led to the confirmation of the nature of both the SSCs and sertoli cells. Results showed that the total number of colonies from day 4 to 11 increased significantly in all groups، independent of CSF1 concentration. In addition، the total number and total area of colonies were higher (not significant) in 10 and 50 ng/mL CSF1 treatments than the control and 100 ng/mL CSF1 groups in all the three evaluations during the experiment. However، this difference was only significant (p<0. 05) between the total area of colonies in the control and 10 ng/mLCSF1 groups at day 4 of co-culture. It was concluded that CSF1 can be a suitable growth factor for improving SSCs colonization in vitro، particularly during the first days of culture where accompanying sertoli cells still have not proliferated sufficiently to support the propagating spermatogonial cells.

Spermatogonial stem cells (SSCs)، the only adult stem cells capable of transferring genetic information to the next generation، are important in cell biology. The most important applications of these cells include the treatment of infertility، fertility preservation of oncological patients، and the creation of transgenic animals. The aim of this study was to evaluate the possibility of transplantation of frozen-thawed bovine spermatogonial cells to the testis of an azoospermic mouse model and to prove the presence of spermatogonial stem cells among bovine spermatogonial cells isolated and proliferated by methods used in the study. Spermatogonial cells were isolated from 3 to 5 month-old bull calves and then were frozen-thawed. Subsequently، the cells were cultured in the presence of glial cell line-derived neurotrophic factor (GDNF 40 ng/ml) for 2 weeks. The stemness of these cells was evaluated by transplanting them to the testis of azoospermic mouse models. The viability rate of fresh cells and frozen cells after thawing were 87. 4 ± 2. 4 and 65 ± 1. 7، respectively، that corresponds to a significant difference (P < 0. 01). After one month، the mice were euthanized and the histopathological evaluation of their testes with fluorescent microscope showed the colonization of marked bovine SSCs in the mouse seminiferous tubules. In our knowledge، this is the first reported case of xenotransplantation in Iran. The results of this study showed that the xenogeneic transplantation of calf frozen-thawed spermatogonial cells to the mouse testis has been successful and SSCs were present among the transplanted cells.

Heat stress causes reduced fertility and significant economic loss in dairy cattle. To override the suppressive effects of heat stress, various hormonal manipulations have been utilized. The aim of this study was to compare the effect of progesterone (in the form of CIDR) and administration of GnRH after insemination on the conception rate of heat stressed dairy cattle. All cows were inseminated at estrus and were then alternately assigned into three groups on day 5 after artificial insemination (AI): i) GnRH group (n=44) received an IM injection of 500 µg GnRH (GONAbreed, PARNEL, Australia,); ii) CIDR group (n = 44) received a CIDR (EAZI-BREED, Hamilton, NZ, containing 1/9 g progesterone) which was removed after a week; and iii) control group (n = 36), which did not receive any treatment. Conception was diagnosed on day 32-39 after AI by ultrasonography. Conception rate in GnRH, CIDR and control groups were 54.5%, 54.5% and 58.3%, respectively. The results demonstrated that there was no significant difference among the three groups (p >0.05). These treatments had had no statistically different effects on lactation, milk yield, days in milk and number of AI (p>0.05). Conception rates within GnRH and CIDR groups in <150 and >150 days in milk subgroups were 74.4%, 40.7%, 84.6% and 41.9%, respectively and differed statistically significantly (p>0.05). Conception rate within control and CIDR groups among <3 and >3 numbers of AI were 80%, 31.2%, 84.2% and 32%, respectively, which was statistically significant (p>0.05). According to the results of this study, the use of GnRH and CIDR after AI did not improve conception rates of mildly heat stressed dairy cattle

When dairy cattle are subjected to heat stress, reproductive efficiency declines.To override the suppressive effects of heat stress various environmental and hormonal manipulations have been utilized. The aim of the present study was to evaluate whether the administration of GnRH and hCG on day 5 post-insemination would improve pregnancy rate in mild heat stressed dairy cattle (summer months). All cows were inseminated at estrus and then on days 5 after AI, they were alternately assigned into three groups: GnRH group(n=44)received an IM injection (5ml) of GnRH (GONAbreed®,PARNEL, Australia,) hCG group (n=44) received an IM injection (1ml) of hCG (Pregnyl®, Daru Pakhsh, Iran) and control group (n=36) which did not received any treatment.Pregnancy was diagnosed on day 32-39 after AI by ultrasonography.. The difference in CR among three groups were analyzed by using chi-square and fisher test. CR in GnRH, hCG and control groups were 54/5%, 61% and 58/3% respectively, that weren’t significant difference among three groups (P>0.05). CR within GnRH groups in ≤150 and>150 days open subgroups were 72%, 40% that is statistically significant (P<0.05) and also CR between GnRH and hCG groups based on Open days>15o was 40%, 76% that is statistically significant (P<0.05). CR within control and hCG groups among <3 and≥3number of AI were 80%, 31%, 77% and 39%respectively, that is statistically different(P<0.05). According to the results of this study, the use of hCG after AI in repeat breeder dairy cows in summer months is recommended.